[Construction of a site-directed mutant of FALL-39 and antibacterial activity of its E. coli-based product]

Constructing a point-directed mutant of FALL-39 and prokaryotic expressive vector, and determining its antibacterial activity.

A two-step polymerase chain reaction (PCR) was used for the site-directed mutagenesis. Two sets of primers (P1, P2, P3, P4) were designed according to FALL-39 gene sequence and mismatch was introduced into P2 for a substitution of AAG for AAT at position 32. Mutagenesis was performed in a two-step PCR, the amplified fragments from the second PCR, which contain the mutation site, were subcloned into the vector PGEX lambda 1T and verified by sequencing analysis. Antibacterial activities of the E. coli-based product against E. coli and P. aeruginosa were detected by using minimal inhibitory concentration (MIC), minimal effective concentration (MEC) and minimal bactericidal concentration (MBC).

A mutant of FALL-39, whose AAT at position 32 is substituted by AAG, was obtained by the two-step PCR. The antibacterial activities of the E. coli-based product of the recombinant FALL-39-Lys32 against E. coli ML-35p and P. aeruginosa ATCC27853 were much stronger than those of FALL-39. MIC and MBC of the FALL-39 against E. coli were 18.75 micrograms/ml and 37.50 micrograms/ml, whereas those of FALL-39-Lys32, 10.94 micrograms/ml and 21.88 micrograms/ml respectively.

A point-directed FALL-39-Lys32 mutant was obtained by using two-step PCR and its antibacterial activity was increased, suggesting that increased cationic FALL-39 mutant might enhance its antibacterial activity.